Neisseria gonorrhoeae

 Neisseria is the only pathogenic Gram negative diplococci 

Two species of Neisseria causes disease in humans; Neisseria meningitidis and Neisseria gonorrhoeae.

 

Neisseria gonorrhoeae (Gonococcus) causes the venereal disease gonorrhoea, the second most common sexually transmitted disease (STDs) of worldwide importance (Chlamydial infections are more common).

 

Human beings are only known hosts of N. gonorrhoeae - It causes natural infection only in humans.

 

Cause an acute, infectious, sexually transmitted disease of the mucous membranes of the genitourinary tract, eye, rectum, and throat - gonorrhea, neonatal conjunctivitis (ophthalmia neonatorum) and pelvic inflammatory disease (PID).

 

Neisseria gonorrhoeae thrives in a CO₂ environment, therefore, the urethra, cervix, rectum, and throat are the main sites of infection.

 

Gonococcus was first described in gonorrheal pus by Neisser in 1879.

Bumm in 1885 cultured the coccus & proved its pathogenicity by inoculating human volunteers.

 

Morphology:

·         Gram-negative, oxidase-positive, diplococcus (seen in pairs) – typically kidney shaped -adjacent sides concave-

·         In urethral discharge it is predominately found within the polymorphs - Intracellular - Some cells may contain as many as 100 cocci.

Urethral exudate containing Neisseria gonorrhoeae from a patient with gonococcal urethritis -typical intracellular gram-negative diplococci, and extracellular gram-negative organisms, which is diagnostic for gonococcal urethritis.

·         Nonmotile

·         Non-capsulated

·         Have pili- facilitate adhesion of the cocci to mucosal surface & promote virulence 

 

Cultural characteristics

• ·         More difficult to grow
• ·         Aerobic -may grow anaerobically also
• ·         Growth occurring best at pH 7.2 – 7.6 
• ·         Optimum temperature for growth – 35 - 36⁰ C; no growth if the temperature is less than 25⁰C or more than 38.5⁰C
• ·         Growth is good in presence of 5 -10 % CO₂
• ·         Grow well on enriched media like Chocolate Agar
• ·         Selective medium – THAYER – MARTIN medium (contains Vancomycin + Colistin + Nystatin which inhibit most of the contaminants like nonpathogenic Neisseria).
• ·         Small, round, translucent, convex or slightly umbonate with finely granular surface and lobate margins.
• ·         Soft and easily emulsifiable

 

Thayer-Martin agar

Biochemical

• ·         Oxidase positive (prompt positive reaction)
• ·         Catalase positive
• ·         Glucose  utilized with acid production, but not maltose, sucrose/lactose
• ·         Indole  not produced
• ·         Nitrates not reduced

Antigenic/Virulence Factors

 

•  Pili - Are hair like structures extending from the surface - made up of pilin proteins -                     -Pilin proteins are antigenically different in almost all strains- A single strain can produce several antigenically distinct pili. Piliated gonococci are usually virulent, whereas non-piliated strains are avirulent 

• Lipooligosaccharide – Outer membrane contains LOS-lipooligosaccharide (endotoxin) – responsible for toxicity 

• Outer membrane Proteins – many different proteins 

• Protein I (por) – Forms pore on surface. Each strain expresses one type of protein I. It helps in serotyping of gonococci. Two variants of protein I – IA & IB. Any one strain carries either IA or IB but not both. 24 serovars of type IA & 32 serovars of type IB. 
• Protein II (opa) – Opacity associated outer membrane protein (OPA). Help in attachment to host cell. Strains with OPA protein form opaque colonies.  
• Protein III – is associated with protein I in the formation of pores on the cell surface &  plays a role in the exchange of molecules across the outer membrane.

• IgA1 protease - The main host defenses against gonococci are antibodies (IgA and IgG), complement, and neutrophils. • IgA protease degrades and inactivates IgA which plays a major role in mucosal defense. 

•  Transferrin (Iron binding proteins) specifically bind and internalize iron from host‐derived proteins, including transferrin, lactoferrin

•  Plasmids-Gonococci contains several cryptic plasmids –  transmissible plasmids contain genes that code for beta lactamase which causes resistance to penicillin. 
• 
  

Resistance

• Very delicate organism - Readily killed by drying, heat & antiseptic 
• Strict parasite & dies in 1 – 2 hours in exudate outside the body. 
• In culture, the coccus dies in 3 – 4 days but survives in slant culture at 35⁰C if kept under sterile paraffin oil. 
• Cultures – preserved for years if frozen quickly & stored at – ^- 70⁰C.

Epidemiology

• ·   Exclusively a human disease- no natural infection in animals. Experimental infection in chimpanzees (urethral inoculation)  and mice (intracerebral inoculation)
• ·         Humans-only source of infection
• ·         Asymptomatic carriage in women- major factor in spreading of infection
• ·         Fomites do not transmit the disease
• ·         The only non-venereal infection is Ophthalmia neonatorum/conjunctivitis of the newborn.- once very common, now controlled by the practice of administering 1% silver nitrate solution into the eyes of all newborns

 Mode of transmission

1.      Sexual transmission: Acquired during unprotected sex with infected partner.

2.    Neonates acquire Neisseria gonorrhoeae from mother during passage through the birth canal.  In newborn infants, Neisseria gonorrhoeae causes Ophthalmia neonatorum (purulent conjunctivitis).

The name Gonorrhea is derived from Greek words- Gonos (seed) rhoia (flow) - Describes a condition in which semen flows from the male organ without erection

 

Pathogenesis 

• Causes disease only in humans. Transmitted sexually both in males and females.

    Once inside the body the gonococci attach to the microvilli of mucosal cells by means of pili and protein II, which function as adhesins.  This attachment prevents the bacteria from being washed away by normal cervical and vaginal discharges or by the flow of urine  They are then phagocytosed by the mucosal cells and  transported through the intercellular spaces and subepithelial tissue. 

Incubation period of 2-8 days.

Phagocytes, such as neutrophils, also may contain gonococci inside vesicles. Because the gonococci are intracellular at this time, the host’s defenses have little effect on the bacteria. Following penetration of the bacteria, the host tissue responds locally by the accumulation of mast cells, more PMNs (polymorphonuceleocytes), and anitbody-secreting plasma cells. These cells are later replaced by fibrous tissue that may lead to urethral closing, or stricture, in males

Gonococci causes both localized infections, usually in the genital tract, and disseminated infections – Mainly 

1. Gonorrhea & Pelvic inflammatory disease (PID) - Venereal

2. Neonatal conjunctivitis (ophthalmia neonatorum) - Non-venereal

Gonorrhea  

Gonorrhoea in men is characterized primarily by urethritis with mucopurulent discharge containing gonococci in large numbers, accompanied by dysuria (painful urination). Chronic urethritis lead to stricture formation. Other complications include epididymitis, prostatitis (painful condition that involves inflammation of the prostate), peri-urethral abscesses and “water can perineum” with multiple discharging sinuses.

:

(Epididymitis - inflammation of the epididymis, which is a tube located at the back of the testicles that stores and carries sperm

Stricture -narrowing of the urethra which restricts or slows the flow of urine in)

 

Gonorrhoea in women, infection is located primarily in the urethra and endocervix (cervicitis), causing a purulent vaginal discharge and inter-menstrual bleeding. Vaginal mucosa is not usually affected due to acidic pH. Infection may extend to Bartholin’s glands ( glands located near the opening of the vagina which secrete mucus to lubricate the vagina), endometrium and fallopean tubes.  The most frequent complication in women is an ascending infection of the uterine tubes (Salphingitis (inflammation of the fallopian tubes) and Pelvic Inflammatory Disease), which can result in sterility or ectopic pregnancy as a result of scarring of tissues.

Rarely, peritonitis and peri-hepatic inflammation is seen (Fitz-Hugh-Curtis syndrome). Also, Proctitis (inflammation of the anus and the lining of the rectum), conjunctivitis (due to autoinoculation) and rarely, blood invasion leading to arthritis, ulcerative endocarditis, or meningitis

 Clinical disease is less severe in women -Asymptomatic carriage common in women. Asymptomatic carriage rare in men.

 

Disseminated infections- Disseminated gonococcal infections occurs via the blood stream.  Septicemia, infections of skin and joints in 1-3 % of women (arthralgia/ arthritis) - more common in women due to untreated symptomatic infections.

 

Non-venereal infections - In newborns, gonococcal ophthalmia in the newborn, due to direct infection during passage through birth canal (vertical transmission)

Ophthalmia neonatorum/ gonococcal ophthalmia - An eye infection which may develop within 2/3 days of vaginal delivery, affects cornea and can cause blindness, purulent conjunctivitis, acquired at delivery. This was once a leading cause of blindness in many parts of the world (now controlled by the practice of administering 1% silver nitrate solution into the eyes of all newborns)

 

 

Opthalmia neonatorum

 

  

Laboratory diagnosis of Gonorrhoea

 Sample: Urethral/Cervical/Vaginal discharge

§  To obtain a urethral specimen, swab is inserted approximately 2cm in urethra and rotated gently before withdrawing.

§  If there is profuse urethral discharge in male, it can be collected without inserting the swab.

§  A few drops of first voided urine can be used in males, but the sensitivity is low compared to discharge.

Transport: Swabs collected for isolation of gonococci may be transported to the laboratory in modified Staurt’s or Amie’s charcoal transport media and held at room temperature until inoculated to culture media. Good recovery of gonococci is possible if swabs are cultured within 12 hours of collection.

 

 

• Gram Staining

For men, a gram-stained smear of urethral discharge (exudate) showing intracellular Gram-negative diplococci is diagnostic.

Women may carry normal vaginal flora such as Veillonella or occasional gram-negative coccobacilli, may resemble gonococci. In case of women use of fluorescent antibody techniques for identification to increase sensitivity and microscopy for specifity.

• Culture

Specimens inoculated on a pre-warmed plate, immediately after collection, if not possible, collect on charcoal impregnated swabs and transport to laboratory on appropriate medium.

In acute gonorrhea, Chocolate agar/Mueller Hinton agar inoculated with the sample, incubation at 35-36^oC, under 5-10% CO₂

In chronic cases, where mixed infection can be seen, use selective media like,  Thayer Martin Medium {Chocolate agar containing antibiotics - vancomycin, colistin, trimethoprim, and nystatin or  Modified Newyork City Medium (MNC) .

 

 Biochemical tests for Neisseria gonorrhoeae identification 

• ·         Oxidase Test: Positive
• ·         Ferments glucose but not maltose, sucrose or lactose
• ·         DNase Test: Negative
• ·         Beta-galactosidase (ONPG) Test: Negative

Serological Tests

• Precipitation, complement fixation test, passive agglutination, immunofluorescence, radioimmunoassay etc-
• Not useful for diagnosis- it becomes positive only some weeks after infection is established, can remain positive for months/years afterwards. Can show positive with meningococcal infection.
• Enzyme-linked immunosorbent assay (ELISA) is also used as a rapid test and is sensitive to gonorrhea.

Molecular Diagnosis:

• PCR method or Nucleic Acid Amplification Tests (NAATs) to detect the presence of gonococcal nucleic acids in patient specimens- highly sensitive and specific.
• Gonorrhea nucleic acid amplification (NAAT) testing-detects DNA of the gonococci and is considered the optimal test for gonorrhea infection- on a urine sample or a swab taken from a site of potential infection

 Treatment

• Initially sulphonamides and penicillin but there was rapid development and spreading of drug resistance especially by Penicillinase producing Neisseria gonorrhoeae.
• Now, Cefixime/Ceftriaxone or Ciprofloxacin with  Doxycycline treatment for 7 days/Erythromycn single oral dose

Prophylaxis

The prevention of gonorrhea involves the use of safety measures and the immediate treatment of symptomatic patients and their contacts.

• ·         Early detection of cases
• ·         Contact tracing
• ·         Health education
• ·         No vaccination (no immunity even by clinical infection)